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1.
Nat Commun ; 11(1): 3888, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753666

RESUMO

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive. The co-localization of histones with antimicrobial peptides (AMPs) in immune cells suggests that histones may be part of a larger antimicrobial mechanism in vivo. Here we report that histone H2A enters E. coli and S. aureus through membrane pores formed by the AMPs LL-37 and magainin-2. H2A enhances AMP-induced pores, depolarizes the bacterial membrane potential, and impairs membrane recovery. Inside the cytoplasm, H2A reorganizes bacterial chromosomal DNA and inhibits global transcription. Whereas bacteria recover from the pore-forming effects of LL-37, the concomitant effects of H2A and LL-37 are irrecoverable. Their combination constitutes a positive feedback loop that exponentially amplifies their antimicrobial activities, causing antimicrobial synergy. More generally, treatment with H2A and the pore-forming antibiotic polymyxin B completely eradicates bacterial growth.


Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Estruturas Cromossômicas/efeitos dos fármacos , Histonas/metabolismo , Prótons , Animais , Estruturas Cromossômicas/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Imunidade Inata , Mamíferos , Polimixina B/farmacologia , Análise de Sequência de RNA , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
2.
Nat Commun ; 11(1): 4226, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839450

RESUMO

Intercellular signaling is indispensable for single cells to form complex biological structures, such as biofilms, tissues and organs. The genetic tools available for engineering intercellular signaling, however, are quite limited. Here we exploit the chemical diversity of biological small molecules to de novo design a genetic toolbox for high-performance, multi-channel cell-cell communications and biological computations. By biosynthetic pathway design for signal molecules, rational engineering of sensing promoters and directed evolution of sensing transcription factors, we obtain six cell-cell signaling channels in bacteria with orthogonality far exceeding the conventional quorum sensing systems and successfully transfer some of them into yeast and human cells. For demonstration, they are applied in cell consortia to generate bacterial colony-patterns using up to four signaling channels simultaneously and to implement distributed bio-computation containing seven different strains as basic units. This intercellular signaling toolbox paves the way for engineering complex multicellularity including artificial ecosystems and smart tissues.


Assuntos
Comunicação Celular/genética , Biologia Computacional/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Mutação , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 11(1): 4045, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792484

RESUMO

Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.


Assuntos
Luz , Optogenética/métodos , Cromatografia de Afinidade , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ligação Proteica/efeitos da radiação , Proteínas/química , Proteínas/metabolismo
4.
Nat Commun ; 11(1): 3834, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737309

RESUMO

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.


Assuntos
Escherichia coli/efeitos dos fármacos , Optogenética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Tetraciclina/farmacologia , Tetraciclinas/farmacologia , Transcrição Genética/efeitos dos fármacos , Clonagem Molecular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fotólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Raios Ultravioleta
5.
Nat Commun ; 11(1): 3841, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737323

RESUMO

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Indóis/química , Proteínas Repressoras/química , Vorinostat/química , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/metabolismo , Indóis/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Termodinâmica , Vorinostat/metabolismo
6.
Nat Commun ; 11(1): 4072, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792663

RESUMO

Cpf1-linked base editors broaden the targeting scope of programmable cytidine deaminases by recognizing thymidine-rich protospacer-adjacent motifs (PAM) without inducing DNA double-strand breaks (DSBs). Here we present an unbiased in vitro method for identifying genome-wide off-target sites of Cpf1 base editors via whole genome sequencing. First, we treat human genomic DNA with dLbCpf1-BE ribonucleoprotein (RNP) complexes, which convert C-to-U at on-target and off-target sites and, then, with a mixture of E. coli uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, which removes uracil and produces single-strand breaks (SSBs) in vitro. Whole-genome sequencing of the resulting digested genome (Digenome-seq) reveals that, on average, dLbCpf1-BE induces 12 SSBs in vitro per crRNA in the human genome. Off-target sites with an editing frequency as low as 0.1% are successfully identified by this modified Digenome-seq method, demonstrating its high sensitivity. dLbCpf1-BEs and LbCpf1 nucleases often recognize different off-target sites, calling for independent analysis of each tool.


Assuntos
Citidina/metabolismo , Endonucleases/metabolismo , Sequenciamento Completo do Genoma/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citidina/genética , DNA/genética , DNA/metabolismo , Endonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Genoma Humano/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Guia/genética
7.
Nat Commun ; 11(1): 3958, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769971

RESUMO

Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.


Assuntos
Dimetilaliltranstransferase/metabolismo , Liases Intramoleculares/metabolismo , Alternaria/enzimologia , Domínio Catalítico , Dimetilaliltranstransferase/química , Ensaios Enzimáticos , Escherichia coli/metabolismo , Fusarium/enzimologia , Indóis/química , Indóis/metabolismo , Liases Intramoleculares/química , Cinética , Ligantes , Modelos Moleculares , Prenilação , Terpenos/metabolismo
8.
PLoS Comput Biol ; 16(8): e1008125, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776925

RESUMO

In the growing field of metabolic engineering, where cells are treated as 'factories' that synthesize industrial compounds, it is essential to consider the ability of the cells' native metabolism to accommodate the demands of synthetic pathways, as these pathways will alter the homeostasis of cellular energy and electron metabolism. From the breakdown of substrate, microorganisms activate and reduce key co-factors such as ATP and NAD(P)H, which subsequently need to be hydrolysed and oxidized, respectively, in order to restore cellular balance. A balanced supply and consumption of such co-factors, here termed co-factor balance, will influence biotechnological performance. To aid the strain selection and design process, we used stoichiometric modelling (FBA, pFBA, FVA and MOMA) and the Escherichia coli (E.coli) core stoichiometric model to investigate the network-wide effect of butanol and butanol precursor production pathways differing in energy and electron demand on product yield. An FBA-based co-factor balance assessment (CBA) algorithm was developed to track and categorise how ATP and NAD(P)H pools are affected in the presence of a new pathway. CBA was compared to the balance calculations proposed by Dugar et al. (Nature Biotechnol. 29 (12), 1074-1078). Predicted solutions were compromised by excessively underdetermined systems, displaying greater flexibility in the range of reaction fluxes than experimentally measured by 13C-metabolic flux analysis (MFA) and the appearance of unrealistic futile co-factor cycles. With the assumption that futile cycles are tightly regulated in reality, the FBA models were manually constrained in a step-wise manner. Solutions with minimal futile cycling diverted surplus energy and electrons towards biomass formation. As an alternative, the use of loopless FBA or constraining the models with measured flux ranges were tried but did not prevent futile co-factor cycles. The results highlight the need to account for co-factor imbalance and confirm that better-balanced pathways with minimal diversion of surplus towards biomass formation present the highest theoretical yield. The analysis also suggests that ATP and NAD(P)H balancing cannot be assessed in isolation from each other, or even from the balance of additional co-factors such as AMP and ADP. We conclude that, through revealing the source of co-factor imbalance CBA can facilitate pathway and host selection when designing new biocatalysts for implementation by metabolic engineering.


Assuntos
Simulação por Computador , Escherichia coli , Engenharia Metabólica/métodos , Algoritmos , Biomassa , Butanóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Modelos Biológicos
9.
Nat Commun ; 11(1): 4035, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788578

RESUMO

Polyphosphates are linear polymers and ubiquitous metabolites. Bacterial polyphosphates are long chains of hundreds of phosphate units. Here, we report that mouse survival of peritoneal Escherichia coli sepsis is compromised by long-chain polyphosphates, and improves with bacterial polyphosphatekinase deficiency or neutralization using recombinant exopolyphosphatase. Polyphosphate activities are chain-length dependent, impair pathogen clearance, antagonize phagocyte recruitment, diminish phagocytosis and decrease production of iNOS and cytokines. Macrophages bind and internalize polyphosphates, in which their effects are independent of P2Y1 and RAGE receptors. The M1 polarization driven by E. coli derived LPS is misdirected by polyphosphates in favor of an M2 resembling phenotype. Long-chain polyphosphates modulate the expression of more than 1800 LPS/TLR4-regulated genes in macrophages. This interference includes suppression of hundreds of type I interferon-regulated genes due to lower interferon production and responsiveness, blunted STAT1 phosphorylation and reduced MHCII expression. In conclusion, prokaryotic polyphosphates disturb multiple macrophage functions for evading host immunity.


Assuntos
Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Polifosfatos/metabolismo , Animais , Apresentação do Antígeno/imunologia , Polaridade Celular , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon Tipo I/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Fenótipo , Sepse/imunologia , Análise de Sobrevida , Transcriptoma/genética
10.
Nat Commun ; 11(1): 3796, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732900

RESUMO

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Divisão Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo
11.
Nat Commun ; 11(1): 3802, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732903

RESUMO

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Desdobramento de Proteína , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Estresse Mecânico , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , NADP/metabolismo , Transporte Proteico , Proteínas SecA/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
12.
Nat Commun ; 11(1): 3803, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732991

RESUMO

Microbial communities comprised of phototrophs and heterotrophs hold great promise for sustainable biotechnology. Successful application of these communities relies on the selection of appropriate partners. Here we construct four community metabolic models to guide strain selection, pairing phototrophic, sucrose-secreting Synechococcus elongatus with heterotrophic Escherichia coli K-12, Escherichia coli W, Yarrowia lipolytica, or Bacillus subtilis. Model simulations reveae metabolic exchanges that sustain the heterotrophs in minimal media devoid of any organic carbon source, pointing to S. elongatus-E. coli K-12 as the most active community. Experimental validation of flux predictions for this pair confirms metabolic interactions and potential production capabilities. Synthetic communities bypass member-specific metabolic bottlenecks (e.g. histidine- and transport-related reactions) and compensate for lethal genetic traits, achieving up to 27% recovery from lethal knockouts. The study provides a robust modelling framework for the rational design of synthetic communities with optimized growth sustainability using phototrophic partners.


Assuntos
Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Processos Heterotróficos/fisiologia , Processos Fototróficos/fisiologia , Synechococcus/metabolismo , Yarrowia/metabolismo , Aldeídos/metabolismo , Bacillus subtilis/genética , Reatores Biológicos/microbiologia , Escherichia coli/genética , Etanol/metabolismo , Formaldeído/metabolismo , Metanol/metabolismo , Microbiota/fisiologia , Modelos Biológicos , Ácido Succínico/metabolismo , Synechococcus/genética , Yarrowia/genética
13.
BMC Bioinformatics ; 21(1): 308, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664870

RESUMO

BACKGROUND: Inferring gene regulatory networks (GRNs) from gene expression data remains a challenge in system biology. In past decade, numerous methods have been developed for the inference of GRNs. It remains a challenge due to the fact that the data is noisy and high dimensional, and there exists a large number of potential interactions. RESULTS: We present a novel method, namely priori-fused boosting network inference method (PFBNet), to infer GRNs from time-series expression data by using the non-linear model of Boosting and the prior information (e.g., the knockout data) fusion scheme. Specifically, PFBNet first calculates the confidences of the regulation relationships using the boosting-based model, where the information about the accumulation impact of the gene expressions at previous time points is taken into account. Then, a newly defined strategy is applied to fuse the information from the prior data by elevating the confidences of the regulation relationships from the corresponding regulators. CONCLUSIONS: The experiments on the benchmark datasets from DREAM challenge as well as the E.coli datasets show that PFBNet achieves significantly better performance than other state-of-the-art methods (Jump3, GEINE3-lag, HiDi, iRafNet and BiXGBoost).


Assuntos
Algoritmos , Redes Reguladoras de Genes , Área Sob a Curva , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Curva ROC
14.
PLoS Biol ; 18(7): e3000564, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32701952

RESUMO

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved ß-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.


Assuntos
Amiloide/metabolismo , Ervilhas/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/metabolismo , Amiloide/ultraestrutura , Detergentes/farmacologia , Escherichia coli/metabolismo , Íons , Pancreatina/metabolismo , Ervilhas/efeitos dos fármacos , Pepsina A/metabolismo , Agregados Proteicos , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/farmacologia , Proteínas de Armazenamento de Sementes/ultraestrutura
15.
Extremophiles ; 24(5): 733-747, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32699914

RESUMO

The compatible solutes ectoine and hydroxyectoine are synthesized by many microorganisms as potent osmostress and desiccation protectants. Besides their successful implementation into various skincare products, they are of increasing biotechnological interest due to new applications in the healthcare sector. To meet this growing demand, efficient heterologous overproduction solutions for ectoines need to be found. This study is the first report on the utilization of the non-halophilic biosynthesis enzymes from Acidiphilium cryptum DSM 2389T for efficient heterologous production of ectoines in Escherichia coli. When grown at low salt conditions (≤ 0.5% NaCl) and utilizing the cheap carbon source glycerol, the production was characterized by the highest specific production of ectoine [2.9 g/g dry cell weight (dcw)] and hydroxyectoine (2.2 g/g dcw) reported so far and occurred at rapid specific production rates of up to 345 mg/(g dcw × h). This efficiency in production was related to an unprecedented carbon source conversion rate of approx. 60% of the theoretical maximum. These findings confirm the unique potential of the here implemented non-halophilic enzymes for ectoine production processes in E. coli and demonstrate the first efficient heterologous solution for hydroxyectoine production, as well as an extraordinary efficient low-salt ectoine production.


Assuntos
Diamino Aminoácidos , Escherichia coli , Acidiphilium/genética , Diamino Aminoácidos/metabolismo , Escherichia coli/metabolismo , Família Multigênica
16.
Phys Rev Lett ; 125(2): 028103, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32701325

RESUMO

Bacterial ribosomes are composed of one-third protein and two-thirds RNA by mass. The predominance of RNA is often attributed to a primordial RNA world, but why exactly two-thirds remains a long-standing mystery. Here we present a quantitative analysis, based on the kinetics of ribosome self-replication, demonstrating that the 1∶2 protein-to-RNA mass ratio uniquely maximizes cellular growth rates in E. coli. A previously unrecognized growth law, and an invariant of bacterial growth, also follow from our analysis. The growth law reveals that the ratio between the number of ribosomes and the number of polymerases making ribosomal RNA is proportional to the cellular doubling time. The invariant is conserved across growth conditions and specifies how key microscopic parameters in the cell, such as transcription and translation rates, are coupled to cellular physiology. Quantitative predictions from the growth law and invariant are shown to be in excellent agreement with E. coli data despite having no fitting parameters. Our analysis can be readily extended to other bacteria once data become available.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Modelos Biológicos , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Escherichia coli/genética , Ribossomos/genética
17.
Nature ; 584(7821): 470-474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32669712

RESUMO

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Fermentação , Gluconeogênese , Glucose/metabolismo , Glicólise , Metabolômica , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
18.
BMC Bioinformatics ; 21(1): 318, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32690031

RESUMO

BACKGROUND: Gene Regulatory Networks (GRNs) have been previously studied by using Boolean/multi-state logics. While the gene expression values are usually scaled into the range [0, 1], these GRN inference methods apply a threshold to discretize the data, resulting in missing information. Most of studies apply fuzzy logics to infer the logical gene-gene interactions from continuous data. However, all these approaches require an a priori known network structure. RESULTS: Here, by introducing a new probabilistic logic for continuous data, we propose a novel logic-based approach (called the LogicNet) for the simultaneous reconstruction of the GRN structure and identification of the logics among the regulatory genes, from the continuous gene expression data. In contrast to the previous approaches, the LogicNet does not require an a priori known network structure to infer the logics. The proposed probabilistic logic is superior to the existing fuzzy logics and is more relevant to the biological contexts than the fuzzy logics. The performance of the LogicNet is superior to that of several Mutual Information-based and regression-based tools for reconstructing GRNs. CONCLUSIONS: The LogicNet reconstructs GRNs and logic functions without requiring prior knowledge of the network structure. Moreover, in another application, the LogicNet can be applied for logic function detection from the known regulatory genes-target interactions. We also conclude that computational modeling of the logical interactions among the regulatory genes significantly improves the GRN reconstruction accuracy.


Assuntos
Algoritmos , Biologia Computacional/métodos , Escherichia coli/genética , Lógica Fuzzy , Redes Reguladoras de Genes , Genes Reguladores , Modelos Genéticos , Simulação por Computador , Escherichia coli/metabolismo , Perfilação da Expressão Gênica
19.
PLoS Negl Trop Dis ; 14(7): e0008488, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716931

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. CONCLUSION: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.


Assuntos
Doenças do Cão/diagnóstico , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Doenças do Cão/sangue , Cães , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Transcriptoma
20.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
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